Virus structure and proteins
Though the different types of HPV virus differ to a small extent, they all have some characteristics in common. The viruses are all small and nonenveloped, meaning they have no lipid bilayer surrounding their capsid, the protein coat surrounding the genome (Münger et al., 2004; Sinal and Woods, 2005; Greenblatt, 2005). Their capsid is an icosahedron, or a polygon with 20 faces, that is 55-nm in diameter (Münger et al., 2004; Greenblatt, 2005; Sinal and Woods, 2005).
The genome of all HPV viruses is circular (Greenblatt, 2005; Sinal and Woods, 2005) and double stranded, with about 8000 base pairs (de Villiers et al., 2004; Münger et al., 2004; Greenblatt, 2005; Moljin et al., 2005; Sinal and Woods, 2005). The genome has eight open reading frames , which overlap to an extent (Greenblatt, 2005; Moljin et al., 2005) and which code for ten proteins (Sinal and Woods, 2005). The genes for these are divided into an early region containing eight genes, that are expressed in the skin's infected basal cells that have yet to differentiate, and a late region with two genes whose protein products exist only in cells after they have differentiated (Greenblatt, 2005; Sinal and Woods, 2005).
The proteins coded by the late genes, L1 and L2, form the virus's capsid (Moljin et al., 2005; Sinal and Woods, 2005). The proteins coded by the early genes, E1 through E8, commandeer the host cell’s replication machinery for viral replication (Moljin et al., 2005; Sinal and Woods, 2005). The incorrectly named E4 protein is actually a late gene (Greenblatt, 2005) that spurs the cell to produce and release mature virions, viruses capable of existing outside the cell and infecting other hosts (Sinal and Woods, 2005).
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Viral "life cycle"
Though viruses are not actually alive, their development progresses through stages intrinsically linked to the cell cycle of the host cell (Sinal and Woods, 2005; Stern, 2005).
Since the virus's propagation is dependent on its replication by the host's DNA replication machinery, which is only in use when the host's genome is being copied (Münger and Howley, 2002; Rapp and Chen, 1998), it is advantageous for the virus to speed cell division and rid the cell of factors that prevent DNA replication (Rapp and Chen, 1998; Greenblatt, 2005). Unfortunately, this leads to the abrogation of processes that exist to ensure that DNA containing errors is not copied, which can lead to the formation of warts and cancer.
Skin cells in the outermost layer of the epidermis are constantly being lost and replaced by cells in the stratum basale, which divide and move up outward the skin's layers. As they move outward, these cells differentiate and usually withdraw from the cell cycle (Rapp and Chen, 1998; Wu et al., 2003). The viral proteins E6 and E7 from high-risk HPV types prevent cells from differentiating and withdrawing from the cell cycle as they move outward through the cell layers, while those from low risk types do not (Baseman and Koutsky, 2005). Differentiating cells begin to produce more and more HPV-encoded proteins until, when they reach the skin surface, they produce complete virions, mature viruses that can survive outside of the host cell (Greenblatt, 2005; Sinal and Woods, 2005). Virions flake off with the discarded skin cells and can go on to infect other hosts and other areas on the same host (Greenblatt, 2005).
Two distinct activities contribute to human papillomavirus 16 E6's oncogenic potential.
Simonson SJ, Difilippantonio MJ, Lambert PF.
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706, USA.
High-risk human papillomaviruses, such as HPV16, cause cervical cancers, other anogenital cancers, and a subset of head and neck cancers. E6 and E7, two viral oncogenes expressed in these cancers, encode multifunctional proteins best known for their ability to bind and inactivate the tumor suppressors p53 and pRb, respectively. In skin carcinogenesis experiments using E6 transgenic (K14E6(WT)) mice, HPV16 E6 was found to contribute to two distinct stages in skin carcinogenesis: promotion, a step involved in the formation of benign papillomas, and progression, the step involved in the malignant conversion of benign tumors to frank cancer. In this study, we compared the tumorigenic properties of K14E6(WT) mice with those of K14E6(delta146-151) mice, which express a mutant form of E6 that cannot bind a family of cellular proteins known as PDZ domain proteins but retains the ability to inactivate p53. In skin carcinogenesis experiments, the K14E6(delta146-151) transgene failed to contribute to the promotion stage of skin carcinogenesis but retained the ability to contribute to the progression stage. Cytogenetic analysis indicated that, although gains of chromosome 6 are consistently seen in tumors arising on K14E6(WT) mice, they are infrequently seen in tumors arising on K14E6(delta146-151) mice. This observation supports the premise that the nature of cancer development in these two mouse strains is distinct. Based on these studies, we conclude that E6 contributes to cancer through its disruption of multiple cellular pathways, one of which is mediated through its interaction with PDZ domain partners and the other through E6's inactivation of p53.
The ATM/p53 pathway is commonly targeted for inactivation in squamous cell carcinoma of the head and neck (SCCHN) by multiple molecular mechanisms.
Bolt J, Vo QN, Kim WJ, McWhorter AJ, Thomson J, Hagensee ME, Friedlander P, Brown KD, Gilbert J.
Stanley S. Scott Cancer Center, LSU Health Sciences Center, New Orleans, LA, USA.
The ATM/p53 pathway plays a critical role in maintenance of genome integrity and can be targeted for inactivation by a number of characterized mechanisms including somatic genetic/epigenetic alterations and expression of oncogenic viral proteins. Here, we examine a panel of 24 SCCHN tumors using various molecular approaches for the presence of human papillomavirus (HPV), mutations in the p53 gene and methylation of the ATM promoter. We observed that 30% of our SCCHN samples displayed the presence of HPV and all but one was HPV type 16. All HPV E6 gene-positive tumors exhibited E6 transcript expression. We observed 21% of the tumors harbored p53 mutations and 42% of tumors displayed ATM promoter methylation. The majority of tumors (71%) were positive for at least one of these events. These findings indicate that molecular events resulting in inactivation of the ATM/p53 pathway are common in SCCHN and can arise by a number of distinct mechanisms.
Regulation of cell cycles is of key importance in human papillomavirus (HPV)-associated cervical carcinogenesis.
Brenna SM, Syrjanen KJ.
State Health Department, Maternity Hospital Leonor Mendes de Barros, Sao Paulo, Brazil.
*****The rapid progress in molecular biology has allowed the identification of the genes involved in different functions of normal cells and has also improved our understanding of the mechanisms of human carcinogenesis. The human papillomavirus (HPV) is a small double-stranded DNA tumor virus and its genes can manipulate cell cycle control to promote viral persistence and replication. The E6 and E7 proteins of high-risk HPV bind to cell cycle regulatory proteins and interfere with both G1/S and G2/M cell cycle checkpoints much more effectively than the low-risk HPV. The difference between the ability of low and high-risk HPV types to induce immortalization and transformation may well lie in their abilities to interact with the various cell cycle components, resulting in the loss of multiple cell cycle checkpoints, which are important in host genome fidelity, thus potentially resulting in accumulation of genetic abnormalities. Cervical cancer is one of the leading malignancies in women worldwide, with substantial morbidity and mortality. According to current concepts, HPV is recognized as the single most important causal agent in the pathogenesis of this cancer. HPV infection clearly precedes the development of malignancy, while being regularly associated with cervical cancer precursor lesions (all grades of squamous intraepithelial lesions). HPV-infected low-grade squamous intraepithelial lesion (SIL) has three possible outcomes: a) it may regress; b) it can persist; or c) it can make a clinical progression to in situ or invasive carcinoma. It has been well established by prospective cohort studies that the spontaneous regression rate increases in parallel with follow-up duration. In contrast, the clinical progression of lesions usually takes place quite rapidly, i.e. during the first two years from diagnosis. The mechanisms responsible for this divergent clinical behavior of HPV-associated squamous intraepithelial lesions are largely unknown, but currently under intense study in different laboratories worldwide.
Sylvia Michelina Fernandes Brenna; Kari Juhani Syrjänen
Maternity Hospital Leonor Mendes de Barros, State Health Department, São Paulo, Brazil
Correspondence
ABSTRACT
The rapid progress in molecular biology has allowed the identification of the genes involved in different functions of normal cells and has also improved our understanding of the mechanisms of human carcinogenesis. The human papillomavirus (HPV) is a small double-stranded DNA tumor virus and its genes can manipulate cell cycle control to promote viral persistence and replication. The E6 and E7 proteins of high-risk HPV bind to cell cycle regulatory proteins and interfere with both G1/S and G2/M cell cycle checkpoints much more effectively than the low-risk HPV. The difference between the ability of low and high-risk HPV types to induce immortalization and transformation may well lie in their abilities to interact with the various cell cycle components, resulting in the loss of multiple cell cycle checkpoints, which are important in host genome fidelity, thus potentially resulting in accumulation of genetic abnormalities. Cervical cancer is one of the leading malignancies in women worldwide, with substantial morbidity and mortality. According to current concepts, HPV is recognized as the single most important causal agent in the pathogenesis of this cancer. HPV infection clearly precedes the development of malignancy, while being regularly associated with cervical cancer precursor lesions (all grades of squamous intraepithelial lesions). HPV-infected low-grade squamous intraepithelial lesion (SIL) has three possible outcomes: a) it may regress; b) it can persist; or c) it can make a clinical progression to in situ or invasive carcinoma. It has been well established by prospective cohort studies that the spontaneous regression rate increases in parallel with follow-up duration. In contrast, the clinical progression of lesions usually takes place quite rapidly, i.e. during the first two years from diagnosis. The mechanisms responsible for this divergent clinical behavior of HPV-associated squamous intraepithelial lesions are largely unknown, but currently under intense study in different laboratories worldwide.
Keywords: Cervical cancers. Cell cycle. Human papillomavirus. Tumor suppressor genes. Histone deacetylase.
RESUMO
O rápido progresso dos estudos em biologia molecular permitiu identificar os genes envolvidos em diferentes funções celulares e também melhorou nossa compreensão sobre os mecanismos da carcinogênese humana. O papilomavírus humano (human papillomavirus, HPV) é um vírus de DNA e os seus genes podem manipular o controle do ciclo celular para promover a sua persistência e replicação. As proteínas E6 e E7 dos HPVs de alto risco oncogênico ligam-se às proteínas reguladoras do ciclo celular e interferem nas fases G1/S e G2/M mais efetivamente do que os HPVs de baixo risco. Os HPVs de baixo e alto risco diferem em sua capacidade de induzir imortalização e transformação celular bem como de interagir com os vários componentes de ciclo celular, o que resulta na perda de pontos de checagem do DNA, importantes para a manutenção do genoma do hospedeiro, e também resulta no acúmulo de anormalidades genéticas. O câncer de colo de útero é um dos principais cânceres genitais em mulheres em todo o mundo, com significativa morbidade e mortalidade. De acordo com conceitos atuais, o HPV é reconhecido como o agente causal mais importante na patogênese deste câncer. A infecção por HPV está associada a todas as lesões intra-epiteliais escamosas do colo do útero. A lesão intra-epitelial escamosa (squamous intraepithelial lesion, SIL) de baixo-grau tem três possíveis resultados: a) pode regredir; b) pode persistir ou c) pode progredir para câncer in situ ou invasivo. Estudos de coorte mostraram que a taxa de regressão espontânea destas lesões aumenta conforme o tempo de seguimento, em contraste com as lesões destinadas a progressão, que normalmente evoluem rapidamente, geralmente nos primeiros dois anos. Os mecanismos responsáveis pelo comportamento clínico da lesão intra-epitelial escamosa associada ao HPV ainda não são totalmente conhecidos, mas atualmente têm sido motivo de estudos em todo o mundo.
Palavras-chave: Câncer cervical. Ciclo celular. Papilomavírus humano. Genes supressores de tumor. Histona deacetilase.
THE CELL CYCLE AND ITS REGULATION
Since the discovery of the deoxyribonucleic acid (DNA) structure, there has been a revolutionary improvement in our knowledge of normal cell functions. The DNA structure is a double-stranded helical molecule composed of two nucleotide chains connected by four nitrogenous bases: adenine (A), thymine (T), guanine (G) and cytosine (C). The DNA code is transmitted when DNA strands are copied during the cell cycle.1 Thus, the replication and division of a cell into genetically identical daughter cells depends on four steps, namely the G1 (gap), S (synthesis), G2 and M (mitosis) phases of the cell cycle. During the G1 phase, the cell accumulates cytoplasmic materials to duplicate the DNA. At the first stop of the cell cycle (named the R checkpoint), checking of the DNA status takes place, before cycle progression. In the event of any abnormality in the genetic information, this must be repaired first, and in such cases cell cycle arrest takes place. In the next steps, named the S and G2 phases, DNA replicates and the materials needed for cell duplication are obtained, respectively. The last step in the cell cycle is called the M phase, in which the cell duplication takes place.1
Cell cycle progression is controlled by a large group of regulatory proteins named cyclin-dependent kinases (CDKs). The active forms of these enzymes only appear in the form of complexes with specific proteins (active in a specific phase of the cycle) known as cyclins. There is often interaction with other proteins such as proliferating cell nuclear antigen (PCNA) and CDK inhibitors. The transitions in the cell cycle take place when the enzymatic activity of a given kinase activates the proteins required for progression from one stage of the cycle to the next. After the division of the cell, the DNA code is transcribed in the nucleus, to messenger ribonucleic acid (mRNA). The latter transfers the genetic information into the cytoplasm, where transfer RNA (tRNA) and synthesis RNA (sRNA) will be responsible for the synthesis of the proteins in the ribosomes. Each cell is programmed for specific functions and finishes its life cycle through apoptosis, the genetic control for removing inappropriate or senescent cells.2
This new understanding of the regulation of normal cell functions has significantly contributed to our concepts of molecular mechanisms in human carcinogenesis. In this review, we give a brief account of the role of human papillomavirus (HPV) as the single most important etiological agent of cervical cancer, by describing the molecular mechanisms whereby this tumor virus interferes with the regulation of the normal cell cycle.
TUMOR SUPPRESSOR GENES
Tumor suppressor genes encode for proteins that regulate cell growth, and prevent the events that lead to malignant transformation of the cells. The first tumor suppressor gene ever cloned was named the Rb gene because it was first identified in retinoblastoma. The Rb gene is located on chromosome 13 and encodes a nuclear protein that regulates gene expression. Loss of the pRb pathway function certainly leads to loss of normal inhibitory controls of the cell cycle progression.1
Another key tumor suppressor gene is the p53 gene, also known as "the guardian of the genome", which is located on the short arm of chromosome 17. This happens to be the most frequently mutated gene in human cancers. The p53 gene was so named because it encodes a 53-kilodalton (kd) nuclear phosphoprotein that is normally present in very low quantities and has a very short half-life in normal cells. When DNA is damaged, however, the p53 gene is activated and the p53 protein interacts with other proteins called CDK/cyclin inhibitors, including the p16, p27 and p21waf1cip1. This concerted action results in the arrest of the cell cycle at the point R, in the G1 phase, to allow the DNA to recover. If the DNA repair is successful, the p21 signals to the CDK/cyclin compound for the cell cycle to continue (Figure 1). In cases where DNA repair is not possible, the p53 protein signals to other regulatory proteins, such as bax, bcl-2 and c-myc, resulting in the induction of apoptosis, which eliminates cells with inappropriate genetic information.1,3 Thus, the p53 is considered to be a checkpoint control factor (Figure 1).
The mutations of the p53 gene have been extensively studied and described in several human malignancies, including cervical cancer.4 In such cases, the p53 gene can lose its functions, e.g. by deletion of one of its alleles (loss of heterozygosity). The cell cycle cannot arrest in the G1/S phase and continued replication of the DNA-damaged cells is allowed, thus leading to genome instability and accumulation of mutations.3,5 The detection of p53 protein using immunohistochemistry has been studied as a prognostic factor in invasive cervical squamous cell carcinoma.6
Polymorphisms of the p53 gene seem to be common and have been described in cervical cancer patients as well. People can carry one of two variations of the p53 gene in codon 72; p53 arg or p53 pro. It has been suggested that HPV oncoprotein (E6) more easily inactivates p53 arg (72) than pro (72), thus bearing some association with the outcome of HPV infections. Indeed, it has been proposed (although not unanimously agreed yet) that people who are homozygous to p53 arg might be less protected against the effects of oncogenic HPV types.7